[Activation of aldehyde dehydrogenase 2 attenuates myocardial injury in diabetic rats by regulating two-pore potassium channel TASK-1].

OBJECTIVE: To investigate the effect of activating aldehyde dehydrogenase 2 (ALDH2) on TASK-1 two-pore potassium channel in myocardial injury of diabetic rats.
 Methods: Diabetic rats were induced by intraperitoneal injection of streptozotocin (55 mg/kg). The diabetic rats were divided into 4 groups: normal group, diabetes at 4th week (DM4W) group, diabetes at 8th week (DM8W) group, and diabetes at 8th week+low concentration of ethanol intervention (DM8W+EtOH) group. The cardiac function of rats was determined by cardiac ultrasonography. The content of hydroxyproline was detected by ELISA. The appearance of myocardial morphous and positive material were observed by HE and PAS staining. The protein expression of TASK-1 was detected by Western blot. Whole-cell patch clamp technique was used to record the action potential duration at 30% and 90% repolarization (APD30, APD90) and two-pore potassium channel TASK-1 current in rat ventricular myocytes. Meanwhile, according to the sensitive electrophysiological characteristics of the potassium channel to acid and base, whether it is two-port potassium channel TASK-1current can be determined.
 Results: Compared with the N group, end-diastole left ventricular diameter (LVIDd), end-systolic left ventricular diameter (LVIDs), hydroxyproline content, TASK-1 protein expression increased, APD30 and APD90 extend, left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF), and TASK-1 current decreased (all P<0.01) in the DM4W group and the DM8W group. HE staining showed that myocardial cell and fiber arrangement disorder, myocyte hypertrophy, myocardial widened and PAS staining reveals that positive material increased in the DM4W group and the DM8W group. Compared with the DM4W group, these changs are more obvious in DM8W rats (P<0.01 or P<0.05). Compared with the DM8W group, in the DM8W+EtOH group, the left ventricular function was restored, the hydroxyproline content and expression of TASK-1 protein were decreased, the TASK-1 current was increased, and APD30 and APD90 were shortened (all P<0.01). HE staining showed that myocardial cell injury was ameliorate and PAS staining showed decreased deposition of positive substances in the DM8W+EtOH group.
 Conclusion: Activation of aldehyde dehydrogenase 2 by low concentration of ethanol can reduce myocardial injury and fibrosis caused by diabetes, and its mechanism may be related to the changes of the two-por potassium channel TASK-1.

LINC00346 regulates NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637 in vascular endothelium injury.

Endothelial injury and dysfunction contributes to atherosclerosis. LINC00346 plays a key role in vascular endothelial cell injury, however, the specific mechanism remains unclear. This study intends to further explore the relationship between LINC00346 and vascular endothelial injury. Circulating LINC00346 was significantly elevated in patients with coronary artery disease and had high diagnostic value for coronary artery disease. In cell experiments, we found that LINC00346 expression was significantly increased in the oxidized low-density lipoprotein (ox-LDL) intervention group, and LINC00346 knockdown delayed ox-LDL induced human umbilical vein endothelial cell (HUVEC) endothelial-to-mesenchymal transition. In addition, knockdown of LINC00346 mitigated ox-LDL-induced NOD-like receptor protein 1 (NLRP1)-mediated inflammasome formation and pyroptosis, but had no significant effect on NLRP3. By observing the number of autophagosome and detecting intracellular autophagic flux, we found that LINC00346 knockdown inhibited the ox-LDL-induced increase in intracellular autophagy level. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA-pull down assay were performed to confirm the inter-molecular interaction. LINC00346 acted as microRNA-637 sponge to up-regulate the expression of NLRP1. Up-regulation of microRNA-637 alleviated NLRP1-mediated pyroptosis in HUVEC and reduced intracellular autophagosome and autolysosome formation. Finally, we explored whether pyropotosis and autophagy interact with each other. We found that inhibition of intracellular autophagy could alleviate NLRP1-mediated pyroptosis. In conclusion, LINC00346 inhibited the activation of NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637, therefore mitigating vascular endothelial injury.

MicroRNA-143-5p modulates pulmonary artery smooth muscle cells functions in hypoxic pulmonary hypertension through targeting HIF-1α.

This paper explores the potential mechanism of microRNA-143-5p regulation effects on pulmonary artery smooth muscle cells (PASMCs) functions in hypoxic pulmonary hypertension (HPH) via targeting HIF-1a, which may offer a new idea for HPH therapy. PASMCs were transfected with mimics control/miR-143-5p mimics or inhibitor control/miR-143-5p inhibitor. We used Western blotting and RT-qPCR to detect the protein and mRNA expressions, CCK-8 assay to detect cellular viability, Annexin V-FITC/PI staining and caspase- 3/cleaved caspase-3 protein to evaluate cellular apoptosis, transwell migration experiment for cellular migration measurement and Dual luciferase reporter gene assay to prove the target of miR-143-5p. Cells under hypoxic condition presented the decreased protein and mRNA expressions of α-smooth muscle actin (SM-α-actin), Myocardin, smooth muscle myosin heavy chain (SMMHC), and smooth muscle-22α (SM22α), Calponin1 and Hypoxia-inducible factor-1α(HIF-1α), the increased cell viability and miR-143-5p level; Overexpression of miR-143-5p obviously reduced vascular smooth muscle-specific contraction marker protein levels and cellular apoptosis, increased cellular migration of PASMCs with hypoxia stimulation; Low-expression of miR-143-5p caused the opposite changes, while co-transfected with Si HIF-1 α blocked the beneficial effects of miR-143-5p inhibition on PASMCs under hypoxia. MicroRNA-143-5p can promote the phenotype conversion, proliferation and migration of pulmonary artery smooth muscle cells under hypoxic condition through direct targeting of HIF-1α.

[Effects of mitochondrial aldehyde dehydrogenase 2 on autophagy-associated proteins in neonatal rat myocardial fibroblasts cultured in high glucose].

OBJECTIVE: To investigate whether autophagy mediates the effects of aldehyde dehydrogenase 2 (ALDH2) on the proliferation of neonatal rat cardiac fibroblasts cultured in high glucose. METHODS: Cardiac fibroblasts were isolated from neonatal (within 3 days) SD rats and subcultured. The fibroblasts of the third passage, after identification with immunofluorescence staining for vimentin, were treated with 5.5 mmol/L glucose (control group), 30 mmol/L glucose (high glucose group), or 30 mmol/L glucose in the presence of Alda-1 (an ALDH2 agonist), daidzin (an ALDH2 2 inhibitor), or both. Western blotting was employed to detect ALDH2, microtubule-associated protein 1 light chain 3B subunit (LC3B) and Beclin-1 in the cells, and a hydroxyproline detection kit was used for determining hydroxyproline content in cell culture medium; CCK- 8 kit was used for assessing the proliferation ability of the cardiac fibroblasts after the treatments. RESULTS: Compared with the control cells, the cells exposed to high glucose exhibited obviously decreased expressions of ALDH2, Beclin-1 and LC3B and increased cell number and hydroxyproline content in the culture medium. Treatment of the high glucose-exposed cells with Alda-1 significantly increased Beclin-1, LC3B, and ALDH2 protein expressions and lowered the cell number and intracellular hydroxyproline content, whereas the application of daidzin resulted in reverse changes in the expressions of ALDH2, Beclin-1 and LC3B, viable cell number and intracellular hydroxyproline content in high glucose-exposed cells. CONCLUSIONS: Mitochondrial ALDH2 inhibits the proliferation of neonatal rat cardiac fibroblasts induced by high glucose, and the effect is possibly mediated by the up-regulation of autophagy-related proteins Beclin-1 and LC3B.

[Changes of two-pore K+ channel TASK-1 in diabetic myocardial injury in rats].

OBJECTIVE: To investigate the changes of the two- pore K+ channel TASK-1 in diabetic rats with myocardial injury. METHODS: Thirty-six SD rats were divided into normal group (N), diabetes at 4 weeks (DM 4W) group, and diabetes at 8 weeks (DM 8W) group. The cardiac functions of the rats were determined using cardiac ultrasonography, and the body weight and heart weight of the rats at different time points were measured to calculate the heart/body weight ratio (HW/BW). Myocardial fibrosis in the rats was assessed using Masson's staining. The protein expression of TASK-1 in the myocardium was detected using Western blotting. Whole- cell patch clamp technique was used to record the action potential duration (APD) and twopore domain potassium channel TASK- 1 current in acutely isolated rat ventricular myocytes. meanwhile, The inhibition of TASK-1 current was observed by the TASK-1 specific inhibitor ML-365. RESULTS: Compared with the normal group, the diabetic rats showed significantly increased HW/BW (P < 0.05), end- diastole left ventricular diameter (LVIDd), end- systolic left ventricular diameter (LVIDs), and TASK-1 protein expression, with obviously decreased left ventricular diameter shortening rate (FS) and ejection fraction (EF) (P < 0.01). Masson staining showed that in diabetic rats, the collagen fibers were thickened, interwoven into a network with uneven arrangement and increased deposition. Compared with DM 4W group, the rats in DM 8W group exhibited progressive increases in LVIDd, LVIDs, HW/BW, and TASK-1 expression (P < 0.01 or 0.05); FS and EF were further decreased (P < 0.01). Masson staining showed worsened morphological changes of the myocardium with increased deposition. Compared with that in the normal group, the current of TASK- 1 in diabetic rats at 8 weeks was significantly reduced (P < 0.01) and the duration of action potential was extended (P < 0.05). The TASK-1 current was successfully inhibited by ML-365. CONCLUSIONS: Diabetes can induce myocardial fibrosis and aggravate myocardial injury possibly in relation to changes in the protein expression and current of the two-port potassium channel TASK-1.

[Effect of low-dose ethanol consumption on expression of nuclear factor-κB in diabetic rats with myocardial injury].

OBJECTIVE: To investigate the effect of low-dose ethanol on the expression of nuclear factor-κB (NF-κB) in diabetic rats with myocardial injury. METHODS: Rat models of diabetes were established by an intraperitoneal injection of 55 mg/kg streptozotocin (STZ). After successful modeling, the rats were given 2.5% ethanol (daily dose of 20 mg/kg) for 1 week, followed by 5% ethanol (daily dose of 39.45 mg/kg) for another 7 weeks. Normal rats without STZ injection and diabetic rats without ethanol treatment serve as the normal control and diabetic model groups, respectively. The ventricular function of the rats was determined using echocardiography. The plasma levels of interleukin-1 (IL-1) and IL-4 were detected in the rats, and the expressions of 4-HNE, NF-κB and IKK proteins in the left anterior myocardium was evaluated using immunohistochemistry or Western blotting; the ultrastructural changes of the myocardium were observed using transmission electron microscopy. RESULTS: Compared with the normal control group, the diabetic rats showed significantly lowered systolic and diastolic functions of the left ventricle, increased plasma level of IL-1 and myocardial 4-HNE expression (P < 0.01), decreased plasma level of plasma IL-4 (P < 0.01), and increased myocardial expressions of NF-κB and IKK proteins (P < 0.01). Transmission electron microscopy revealed myofibrillar rupture, incomplete myofibrillar structure and mitochondrial damage in the cardiac myocytes in the diabetic rats. Compared with the diabetic rats, the rats with low-dose ethanol treatment exhibited improved systolic and diastolic functions of the left ventricle, milder myocardial myofibrillar and mitochondrial damages, and significantly lowered plasma IL-1 level and myocardial expressions of 4-HNE, NF-κB and IKK (P < 0.01), and increased plasma IL-4 level (P < 0.01). CONCLUSIONS: NF-κB expression is increased in the myocardium of diabetic rats with myocardial injury, and low-dose ethanol consumption lowers myocardial expression of NF-κB in diabetic rats, suggesting the involvement of NF-κB signaling pathway in the protective effect of low-dose ethanol against myocardial injury in diabetes mellitus.

[Mitochondrial aldehyde dehydrogenase 2 protects against high glucose-induced injury in neonatal rat cardiomyocytes by regulating CaN-NFAT3 signaling pathway].

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for α-SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca2+ concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein (P < 0.05) and increased expressions of CaN (P < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca2+ concentrations (P < 0.01). Alda-1 treatment significantly lowered Ca2+ concentration (P < 0.05), intracellular CaN content (P < 0.01), and CaN and NFAT3 protein expressions (P < 0.05), and increased ALDH2 protein expression (P < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca2+ concentration (P < 0.01) and intracellular CaN content (P < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (P>0.05) but significantly reduced the expression of NFAT3 protein (P < 0.05). CONCLUSIONS: Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca2+-CaN-NFAT3 signaling pathway.

[Effects of irbesartan on Notch1 signaling pathway in diabetic rats with myocardial injury].

OBJECTIVE: To investigate the effects and mechanisms of irbesartan on myocardial injury in diabetic rats, and to analyze the changes of Notch1 signaling pathway in it. METHODS: Thirty rats were randomly divided into four groups:normal control group (CON, n=6), high calorie group (HC, n=6) and diabetes mellitus group (DM, n=9), irbesartan + diabetes group (Ir + DM, n=9). After modeling 8 weeks later, the body weight ratio and left ventricular weight index were measured and the serum levels of triglyceride (TG) and total cholesterol (TC) were measured by automatic biochemical analyzer. The changes of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in myocardium of rats were determined by the kit and the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 assaciated X protein (Bax) protein in myocardium were detected by immunohistochemistry. The expressions of Notch1, Hes-1 and jagged-1 in myocardium of rats were detected by Western blot. RESULTS: Compared with CON group, the levels of heart weight/body weight (H/B), left ventricular weight index(LVWI) and fasting blood glucose(FBG) in HC group were not significantly changed, while the levels of blood lipids, MDA and Bax were increased significantly, and the expressions of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. Compared with HC group, the levels of H/B, LVWI, FBG, MDA and Bax in DM group were increased significantly, and the levels of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. The expression of H/B, LVWI, Notch1, Hes-1 and Jagged-1 in Ir+DM group were increased, but there was no significant difference between the other indexes. The H/B and LVWI in Ir + DM group were significantly lower than those in DM group, the levels of blood lipid and blood glucose did not change significantly, but the incidence of oxidative stress and apoptosis was reduced. While Notch1, Hes-1, Jagged -1 protein expressions were increased. CONCLUSIONS: Diabetes can induce myocardial injury, and irbesartan has myocardial protective effects through activation of Notch1.

Sex differences in subacute toxicity and hepatic microsomal metabolism of triptolide in rats.

Triptolide, a major active component of Tripterygium wilfordii Hook F (TWHF), has multiple pharmacological activities. However, its clinical use is often limited by its severe toxicity. In the present study, we evaluated the oral toxicity of triptolide in Sprague-Dawley rats for 28 days at the dosages of 0, 200 and 400microg/kg/day, respectively. Significant difference in the toxicity of triptolide at 400microg/kg was found between different sexes. The triptolide-treated female rats showed many abnormalities, including anorexia, diarrhea, leanness, suppression of weight gain and food intake, fatty liver, splenomegaly and atrophy of ovaries. In contrast, no such abnormalities were observed in male rats except for the significant reproductive toxicity. Furthermore, the metabolism of triptolide in liver microsomes from both sexes was investigated by HPLC. A greater rate of triptolide metabolism was observed in male rat hepatic microsomes, suggesting that one of the cytochrome P450s (CYPs) responsible for triptolide metabolism is male-specific or predominant at least. The inhibition experiments with CYP inhibitors showed that CYP3A and CYP2B were mainly involved in the metabolism of triptolide. In addition, since CYP3A2 is a male-predominant form in rats, significant sex difference in the metabolism of triptolide disappeared in vitro after anti-rat CYP3A2 antibody pretreatment. Results suggested that CYP3A2 made an important contribution to the sex-related metabolism of triptolide, which may result in the sex differences in triptolide toxicity.